1. Formation of the Androgen Receptor Transcription Complex
Yongfeng Shang, Molly Myers, Myles Brown 
Molecular Cell 
Volume 9, Issue 3, Pages (March 2002)
DOI: /S (02)
Copyright © 2002 Cell Press Terms and Conditions

2. Figure 1 Histone Acetylation in AR-Mediated Gene Transcription
(A) The effects of trichostatin A (TSA) and HDAC1 on DHT-stimulated gene transcription. LNCaP cells were transfected with 50 ng of a PSA promoter-driven luciferase along with 100 ng or 500 ng of an HDAC1 expression construct. After 48 hr of transfection, cells were treated with 10 nM of DHT or with 0, 50, or 200 nM of TSA for 16 hr, and the luciferase activity was measured. Transfection efficiency was normalized using a β-galactosidase expression construct, and the results represent at least three independent experiments.
(B) Western blot analysis of PSA expression in LNCaP cells under treatment with DHT or DHT plus TSA. LNCaP cells were treated with 10 nM DHT or 10 nM DHT plus 100 nM TSA for different times. Total proteins were extracted from cells, and Western blotting was performed using specific antibodies against PSA.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

3. Figure 2
AR Recruitment in the Enhancer and the Promoter of the PSA Gene in Response to DHT Treatment
(A) Schematic diagram of the PSA gene regulatory region. Solid boxes depict putative AREs, and A/B, C/D, E/F, G/H, and I/J are primer pairs used for amplifying corresponding DNA fragments. The numbers are the positions upstream to the PSA gene transcription start site.
(B) ChIP assays of AR occupancy on the PSA gene regulatory region. LNCaP cells were treated with 10 nM DHT. Soluble chromatin was prepared from formaldehyde-cross-linked and sonicated cell cultures. Specific antibodies against AR were used to immunoprecipitate protein-bound DNA fragments. These fragments were amplified by PCR using primers described in (A).
(C) The size of the genomic DNA fragments from solubilized chromatin as described in (B).
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

4. Figure 3
Distinct Roles of the Enhancer and Promoter in the Assembly of AR Coactivator and Corepressor Complexes
(A) The DHT induction of PSA expression is blocked by bicalutamide in LNCaP cells. LNCaP cells were treated with 10 nM DHT or 10 nM DHT and 5 μM bicalutamide for different times. Total proteins were extracted and Western blotting was performed to detect PSA expression.
(B) ChIP assays reveal differential recruitment of coactivators and corepressors to the PSA promoter and enhancer. LNCaP cells were treated with 10 nM DHT (D), 5 μM bicalutamide (B), or vehicle alone (−) for 1 hr. Soluble chromatin was prepared and immunoprecipitated with antibodies against AR, GRIP1, CBP, NCoR, SMRT, HDAC1, and HDAC2. Final DNA fragments were PCR-amplified using the primer pair A/B to detect promoter binding (right panel) or primer pair G/H to detect enhancer binding (left panel).
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

5. Figure 4
An Essential Role of the p160-CBP/p300 Interface in the Assembly of AR Coactivator Complex
(A) Diagrams of the functional domains of GRIP1, subGRIP1, subGRIP1ΔAD1, and subGRIP1ΔAD2. bHLH, basic helix-loop-helix; PAS, Per-Arnt-Sim; NR box, Nuclear receptor binding motif; CoRNR box, corepressor nuclear receptor binding motif; AD, activation domain.
(B) Deletion of the AD1 in subGRIP1 diminishes bicalutamide-stimulated gene transcription. LNCaP cells were transfected with 500 ng of GRIP1, subGRIP1, subGRIP1ΔAD1 (ΔAD1), or subGRIP1ΔAD2 (ΔAD2). Forty-eight hours after transfection, cells were treated with 10 nM DHT or 5 μM bicalutamide. Luciferase activity was measured and normalized with a β-galactosidase expression construct; the results represent at least three independent experiments.
(C) Diminished AR activity in subGRIP1ΔAD1 transfection is not due to the inability of subGRIP1ΔAD1 to bind to bicalutamide-bound AR on PSA gene promoter. ChIP experiments were performed under the same experimental conditions described in (B) to measure the recruitment of GRIP1 or GRIP1 mutants, CBP, and the acetylation of histone (AcH).
(D) Diminished AR activity in subGRIP1ΔAD1 transfection is not due to the failure of subGRIP1ΔAD1 expression. Western blotting was performed under the same experimental conditions described in (B) to measure the expression of GRIP1 and GRIP1 mutants using GRIP1-specific antibodies.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

6. Figure 5
Induction of Cell Cycle Progression in LNCaP Cells Expressing subGRIP1
LNCaP cells were transfected with GRIP1, subGRIP1, subGRIP1ΔAD1 (ΔAD1), or subGRIP1ΔAD2 (ΔAD2) along with a GFP construct. Forty-eight hours after transfection, cells were treated with 10 nM DHT or 5 μM bicalutamide or vehicle alone for another 16 hr. Cells were then collected and analyzed by flow cytometry. The numbers represent the percentage of GFP-positive cells that are in the S/G2/M phases of the cell cycle after 16 hr treatment with DHT or bicalutamide.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

7. Figure 6
Coordination between the Enhancer and Promoter in the Assembly of the AR Transcription Complex
ChIP assays of the recruitment of GRIP1 and RNA polymerase II and histone acetylation to the promoter (A), enhancer (C), or nonfunctional region between the enhancer and promoter (B) of the PSA gene. LNCaP cells were treated with 10 nM DHT for the times indicated. Soluble chromatin was prepared from formaldehyde-cross-linked and sonicated cell cultures. Specific antibodies against GRIP1, pol II, and acetylated histones (AcH) were used to immunoprecipitate protein-bound DNA fragments. These fragments were amplified by PCR using the primer pairs described in Figure 2A. (D) Northern blot analysis of PSA gene expression. Total RNA was extracted, and Northern blotting was performed using exon 2 of the PSA gene as probe. β-Actin expression was used as the control.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions

8. Figure 7 Model of the AR Transcription Complex
Agonist-bound ARs are recruited to both the enhancer and the promoter of the PSA gene. This is followed by a coordinated and ordered recruitment of p160 proteins, CBP, p300, and RNA polymerase II holoenzyme to form the AR transcription complex.
Molecular Cell 2002 9, DOI: ( /S (02) )
Copyright © 2002 Cell Press Terms and Conditions